Molecular Mining of Follicular Fluid for Reliable Biomarkers of Human Oocyte and Embryo Developmental Competence



The occurrence of an intrafollicular biochemistry that can be used to relate oocyte developmental competence with quantitative and qualitative aspects of specific components of ­follicular fluid aspirated at ovum retrieval has been a goal of research in clinical IVF for decades. As a complex mixture of bioactive molecules, some produced in situ and others serum borne, there is no shortage of candidates for investigation. This chapter focuses on current methods of high-resolution biochemical analysis that can be applied to follicular biochemistry, which candidates may be the most promising in this regard, and specific investigational approaches that may have the greatest potential to fulfill this goal. In particular, emphasis is placed on whether there is a need to know both the target(s) and function(s) of candidate molecules as related to the developmental biology of the oocyte.


Molecular mining of follicular fluid Biomarkers of human oocyte development Microanalytical imaging Genetic and biochemical technologies in reproduction Follicular fluid markers 


  1. 1.
    Balaban B, et al. Alpha scientists in reproductive medicine and ESHRE special interest group of embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26:1270–83.Google Scholar
  2. 2.
    Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod. 2008;14:679–90.PubMedCrossRefGoogle Scholar
  3. 3.
    Scott R, Seli E, Miller L, et al. Noninvasive metabolomic profiling of human embryo culture medium using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study. Fertil Steril. 2008;90:77–83.PubMedCrossRefGoogle Scholar
  4. 4.
    Seli E, Botros L, Sakkas D, Burns D. Noninvasive profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 2008;90:2183–9.PubMedCrossRefGoogle Scholar
  5. 5.
    McKenzie LJ, Pangas SA, Carson SA, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod. 2004;19: 2869–74.PubMedCrossRefGoogle Scholar
  6. 6.
    Cillo F, Tiziana A, Brevini L, et al. Association between human oocyte developmental competence and expression levels of some cumulus genes. Reproduction. 2007;134:645–50.PubMedCrossRefGoogle Scholar
  7. 7.
    Jones G, Cram D, Song B, et al. Novel strategy with potential to identify developmentally competent IVF blastocysts. Hum Reprod. 2008;23:1748–59.PubMedCrossRefGoogle Scholar
  8. 8.
    Michael A. Do biochemical predictors of outcome exist? In: Van Blerkom J, Gregory L, editors. Essential IVF: basic research and clinical applications. Boston: Kluwer Academic; 2004. p. 81–110.Google Scholar
  9. 9.
    Van Blerkom J, Trout S. Oocyte selection in contemporary clinical IVF: do follicular markers of oocyte competence exit? In: Elder K, Cohen J, editors. Human preimplantation embryo selection. London: Informa Press; 2007. p. 301–24.CrossRefGoogle Scholar
  10. 10.
    Edwards R. Causes of early embryonic loss in human pregnancy. Hum Reprod. 1986;1:85–98.Google Scholar
  11. 11.
    Kuliev A, Cieslak J, Llkevitch Y, Verlinsky Y. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod Biomed Online. 2003;6:54–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Kalousek D. Pathogenesis of chromosomal mosaicism and its effect on early human development. Am J Med Genet. 2000;91: 39–45.PubMedCrossRefGoogle Scholar
  13. 13.
    Ambartsumyan G, Clark A. Aneuploidy and early human embryo development. Hum Mol Genet. 2008;17(R1):R10–5.PubMedCrossRefGoogle Scholar
  14. 14.
    Menezo Y, Guerin P. Preimplantation embryo metabolism and embryo interaction with the in vitro environment. In: Elder K, Cohen J, editors. Human preimplantation embryo selection. London: Informa Press; 2007. p. 191–200.CrossRefGoogle Scholar
  15. 15.
    Tam P, Ng T, Mao K. Beta-endorphin levels in preovulatory follicles and the outcome of in vitro fertilization. J In Vitro Fertil Embryo Transf. 1988;5:91–5.CrossRefGoogle Scholar
  16. 16.
    Cioffi JV, Blerkom JA, et al. The expression of leptin and its receptors in preovulatory human follicles. Mol Hum Reprod. 1997;3: 467–72.PubMedCrossRefGoogle Scholar
  17. 17.
    Oosterhuis G, Vermes I, Lambalk C, et al. Insulin-like growth factor (IGF)-1 and IGF binding protein-3 concentration in follicular fluid from human stimulated follicles. Fertil Steril. 1998;90:60–4.Google Scholar
  18. 18.
    Mendoza C, Cremades N, Ruiz-Requena E, et al. Relationship between fertilization results after intracytoplasmic sperm injection, and intrafollicular steroid, pituitary hormone and cytokine concentrations. Hum Reprod. 1999;13:863–8.Google Scholar
  19. 19.
    Mendoza C, Ruiz-Requena E, Ortega E, et al. Follicular fluid markers of oocyte developmental potential. Hum Reprod. 2002;17: 1017–22.PubMedCrossRefGoogle Scholar
  20. 20.
    Michael A, Collins T, Norgate D, Gregory L, et al. Relationship between ovarian cortisol:cortisone ratios and the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET). Clin Endocrinol. 1999;51:535–40.CrossRefGoogle Scholar
  21. 21.
    Sabatini L, Wilson C, Lower A, Al-Shawaf T, Grudzinskas J. Superoxide dismutase activity in human follicular fluid after controlled ovarian hyperstimulation in women undergoing in vitro fertilization. Fertil Steril. 1999;72:1027–34.PubMedCrossRefGoogle Scholar
  22. 22.
    Lee K, Joo B, Na Y, et al. Relationships between concentrations of tumor necrosis factor-alpha and nitric oxide in follicular fluid and oocyte quality. Fertil Steril. 2000;17:222–8.Google Scholar
  23. 23.
    Oyawoye O, Abdel Gadir A, Garner A, et al. Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Hum Reprod. 2003;18:2270–4.PubMedCrossRefGoogle Scholar
  24. 24.
    Antczak M. The synthetic and secretory behaviors (nonsteroidal) of ovarian follicular granulosa cells: parallels to cells of the endothelial lineage. In: Van Blerkom J, Gregory L, editors. Essential IVF: basic research and clinical applications. Boston: Kluwer Academic; 2004. p. 1–42.Google Scholar
  25. 25.
    Ocal P, Aydin S, Cepni I, et al. Follicular fluid concentration of vascular endothelial growth factor, inhibin A and inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol Reprod Biol. 2004;115:194–9.PubMedCrossRefGoogle Scholar
  26. 26.
    Pasqualotto E, Agarwal A, Sharma R, et al. Effect of oxidative stress in follicular fluid on the outcome of assisted reproduction procedures. Fertil Steril. 2004;81:973–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Wunder D, Mueller M, Birkhauser M, Bersinger N. Steroids and protein markers in the follicular fluid as indicators of oocyte quality in patients with and without endometriosis. J Assist Reprod Genet. 2005;22:257–64.PubMedCrossRefGoogle Scholar
  28. 28.
    Wu Y, Chang C, Cai J, et al. High bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and subsequent embryonic development. Hum Reprod. 2007;22: 1526–31.PubMedCrossRefGoogle Scholar
  29. 29.
    Ledee N, Lombroso R, Lombardeli L, Selva J, et al. Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor. Hum Reprod. 2008;23:2001–9.PubMedCrossRefGoogle Scholar
  30. 30.
    Sinclair K, Lunn L, Kwong Y, et al. Amino acid and fatty acid composition of follicular fluid as predictors of in-vitro embryo development. Reprod Biomed Online. 2008;16:859–68.PubMedCrossRefGoogle Scholar
  31. 31.
    Godard NM, Pukazhenthi BS, Wildt DE, Comizzoli P. Paracrine factors from cumulus-enclosed oocytes ensure the successful maturation and fertilization in vitro of denuded oocytes in the cat model. Fertil Steril. 2009;91(5 Suppl):2051–60.PubMedCrossRefGoogle Scholar
  32. 32.
    Revelli A, Delle Piane L, Casano S, et al. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009;7:40–53.PubMedCrossRefGoogle Scholar
  33. 33.
    Rodgers R, Irving-Rodgers H. Formation of the ovarian follicular antrum and follicular fluid. Biol Reprod. 2010;82:1021–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Makabe S, Van Blerkom J. Human female reproduction: ovarian development to early embryogenesis. New York: Taylor and Francis; 2006.Google Scholar
  35. 35.
    Van Blerkom J, Motta P. The cellular basis of mammalian reproduction. Baltimore: Urban and Schwartenberg; 1979.Google Scholar
  36. 36.
    Van Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion. 2011;11:797–813.PubMedCrossRefGoogle Scholar
  37. 37.
    Antczak M, Van Blerkom J. Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod. 1997;3:1067–86.PubMedCrossRefGoogle Scholar
  38. 38.
    Antczak M, Van Blerkom J, Clark A. A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor beta2 sequestration in a subpopulation of human ovarian follicle cells. Hum Reprod. 1997;12:2226–34.PubMedCrossRefGoogle Scholar
  39. 39.
    Monteleone P, Giovanni A, Simi G, et al. Follicular fluid VEGF levels directly correlate with perifollicular blood flow in normoresponder patients undergoing IVF. J Assist Reprod Genet. 2008;25:183–6.PubMedCrossRefGoogle Scholar
  40. 40.
    Barroso G, Barrioneuvo M, Rao O, Graham L, et al. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril. 1999;72:1024–6.PubMedCrossRefGoogle Scholar
  41. 41.
    Mantzoros C, Cramer D, Liberman R, Barbieri R. Predictive value of serum and follicular fluid leptin concentrations during assisted reproductive cycles in normal women and in women with polycystic ovarian syndrome. Hum Reprod. 2000;15:539–44.PubMedCrossRefGoogle Scholar
  42. 42.
    Tsai E, Yang C, Chen S, et al. Leptin affects pregnancy outcome of in vitro fertilization and steroidogenesis of human granulosa cells. J Assist Reprod Genet. 2002;19:169–76.PubMedCrossRefGoogle Scholar
  43. 43.
    DePlacido G, Alviggi C, Clariza R, et al. Intra-follicular leptin concentration as a predictive factor for in vitro oocyte fertilization in assisted reproductive techniques. J Endocrinol Invest. 2006;29:719–26.Google Scholar
  44. 44.
    Hazout A, Bouchard P, Seifer D, et al. Serum anti-mullerian hormone/mullerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol. Fertil Steril. 2004;82:1323–9.PubMedCrossRefGoogle Scholar
  45. 45.
    Silberstein T, MacLaughlin D, Shari I, et al. Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predicts both ovarian reserve and embryo morphology. Hum Reprod. 2006;21:159–63.PubMedCrossRefGoogle Scholar
  46. 46.
    Eldar-Geva T, Ben-Chetrit A, Spitz I, et al. Dynamic assays of inhibin B, anti-mullerian hormone and estradiol following FSH stimulation and ovarian ultrasonography as predictors of IVF outcome. Hum Reprod. 2005;20:3178–83.PubMedCrossRefGoogle Scholar
  47. 47.
    Bayer S, Armant D, Dlugi A, Seibel M. Spectrophotometric absorbance of follicular fluid: a predictor of oocyte fertilizing capability. Fertil Steril. 1988;49:442–6.PubMedGoogle Scholar
  48. 48.
    Aharoni A, Ric de Vos C, Verhoeven H, et al. Nontargeted metabolome analysis by use of Fourier transform ion cyclotron mass spectrometry. OMICS. 2002;6:217–34.PubMedCrossRefGoogle Scholar
  49. 49.
    Singh R, Sinclair K. Metabolomics: approaches to assessing oocyte and embryo quality. Theriogenology. 2007;68 Suppl 1:S56–62.PubMedCrossRefGoogle Scholar
  50. 50.
    Piñero-Sagredo E, Nunes S, de Los Santos MJ. NMR metabolic profile of human follicular fluid. NMR Biomed. 2010;23(5):485–95.PubMedCrossRefGoogle Scholar
  51. 51.
    Gregory L. Peri-follicular vascularity: a marker of follicular heterogeneity and oocyte competence and a predictor of implantation in assisted conception cycles. In: Van Blerkom J, Gregory L, editors. Essential IVF: basic research and clinical applications. Boston: Kluwer Academic; 2004. p. 59–80.Google Scholar
  52. 52.
    Van Blerkom J. An overview of determinants of oocyte and embryo developmental competence: specificity, accuracy and applicability in clinical IVF. In: Gerris J, Racowsky C, et al., editors. Single embryo transfer. New York: Cambridge University Press; 2009. p. 17–52.Google Scholar
  53. 53.
    Van Blerkom J. Follicular influences on oocyte and embryo competence. In: De Jonge C, Barratt C, editors. Assisted reproductive technology. Cambridge: Cambridge University Press; 2002. p. 81–105.Google Scholar
  54. 54.
    Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod. 1997;12(5):1047–55.PubMedCrossRefGoogle Scholar
  55. 55.
    Semenza G. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999;15:551–78.PubMedCrossRefGoogle Scholar
  56. 56.
    Bruick R. Oxygen sensing in the hypoxic response pathway: regulation of hypoxia-inducible transcription factor. Genes Dev. 2003;17:2614–23.PubMedCrossRefGoogle Scholar
  57. 57.
    Shrestha S, Costello M, Sjoblom P, et al. Power Doppler ultrasound assessment of follicular vascularity in the early follicular phase and its relationship with outcome in in vitro fertilization. J Assist Reprod Genet. 2006;23:1610169.CrossRefGoogle Scholar
  58. 58.
    Robson S, Barry M, Norman R. Power Doppler assessment of follicular vascularity at the time of oocyte retrieval in in vitro fertilization cycles. Fertil Steril. 2008;90:2179–82.PubMedCrossRefGoogle Scholar
  59. 59.
    Malamitsi-Puchner A, Sarandakou A, Baka S, et al. Concentrations of angiogenic factors in follicular fluid and oocyte-cumulus complex culture medium from women undergoing in vitro fertilization: association with oocyte maturity and fertilization. Fertil Steril. 2001;76:98–101.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Molecular, Cellular and Developmental BiologyUniversity of ColoradoBoulderUSA

Personalised recommendations